1. Field of the Invention
This invention relates to purified human GMP synthetase, its production by recombinant technology and nucleic acid sequences encoding for human GMP synthetase and its use in assaying for inhibitors of human GMP synthetase activity and the inhibitors identifiable by such assays.
Human guanosine 5'-monophosphate (GMP) synthetase is a key enzyme in the de nova synthesis of guanine nucleotides. Biosynthesis of guanine nucleotides is not only essential for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) synthesis, but also for providing guanosine 5'-triphosphate (GTP) which is involved in a number of cellular processes important for cell division. GTP hydrolysis is required for microtubule assembly, protein glycosylation, synthesis of adediine nucleotides, protein translation and activation of G proteins. A key step in the de novo synthesis of guanine nucleotides is the conversion of inosine 5'-monophosphate (IMP) to guanosine 5'-monophosphate (GMP). Two enzymes are involved in converting IMP to GMP: IMP dehydrogenase (EC 1.1.1.205) which catalyzes the oxidation of IMP to XMP (xanthine 5'-monophosphate) and GMP synthetase (EC 6.3.5.2.) which catalyzes the amination of XMP to GTP. The reaction catalyzed by GMP synthetase is: EQU XMP+ATP+glutamine.fwdarw.GMP+AMP+PP.sub.i +glutamate
Both GMP synthetase and IMP dehydrogenase exhibit elevated levels of activity in rapidly proliferating cells, such as neoplastic and regenerating tissues. Inhibition of GMP synthetase or IMP dehydrogenase has been shown to result in the inhibition of cell growth. Because of this anti-proliferative effect of GMP synthetase and IMP dehydrogenase inhibitors, both enzymes are potential targets for anti-cancer and immunosuppressive therapies. In fact, in recent clinical trials, a potent inhibitor of IMP dehydrogenase, mycophenolic acid, was shown to be effective in treatment of transplant rejection and rheumatoid arthritis.
The inhibition of lymphocyte proliferation by mycophenolic acid is closely correlated with the lowering of the intracellular pool of guanine nucleotides. Furthermore, this inhibition of proliferation can be blocked by the addition of exogenous guanosine. These data suggest that the immunosuppressive activity of mycophenolic acid is a result of the depletion of the guanine nucleotide pool. Since GMP synthetase is crucial for the de novo synthesis of guanine nucleotides, the inhibition of GMP synthetase should also result in the depletion of guanine nucleotides and therefore could be a target for immunosuppressive and cancer therapies.
Despite its significance, the information available for human GMP synthetase is limited. Heretofore, the human enzyme has not been obtained in a purified state. Even though a partially purified preparation from human fibroblast cells (T. Page, B, Bakay and W. L. Nyhan; Int. J. Biochem., 16, 117-120, (1984)) has been reported, it contained varying amounts 5'-nucleotidase, purine nucleoside phosphorylase, and GMP kinase. The degree of purification of the enzyme was approximately 50-fold over the naturally occurring material and its specific activity was reported to be 5990 pmoles guanine compound produced/min/mg. By contrast, the present invention discloses a purified human GMP synthetase purified 936-fold over the naturally occurring material with a specific activity of at least 2,500,000 pmoles adenosine 5'-monophosphate (AMP) produced/min/mg. GMP synthetases of mammalian origin have been isolated in varying degrees of purity from a variety of sources. However, mammalian GMP synthetases, even if available in a high degree of purity, are of limited utility in screening for inhibitors for the purposes of human therapy.
Genetic information for human GMP synthetase is not available. Although the cDNA for GMP synthetase has been isolated from E. coli, B.subtilis and D. discoideum, no human or mammalian cDNAs or genes for GMP synthetase are available. Therefore it has not been possible to produce human GMP synthetase by recombinant DNA technology.
Accordingly, human GMP synthetase has not been available in a form suitable either for elucidation of its mechanism or for its use in screening for potential inhibitors. Neither has there been sequence information such as, for example, cDNA sequences coding for human GMP synthetase which would enable the production of human GMP synthetase by recombinant DNA technology. For these reasons, it is desirable to produce pure, well characterized human GMP synthetase in large quantities for the purposes, for example, of elucidating the mechanisms of its activity, identifying its role in various disease states and screening for therapeutically significant inhibitors of guanine nucleotide biosynthesis. Furthermore, it would be desirable to produce human GMP synthetase by means of recombinant DNA technology because it would make the enzyme available in much larger amounts than is practical in purification from natural sources.